Pathogens detected from patients with acute respiratory infections negative for SARS-CoV-2, Saitama, Japan, 2020

Objective During the coronavirus disease pandemic in Japan, all patients with respiratory symptoms were initially tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study describes the respiratory pathogens detected from patients who tested negative for SARS-CoV-2 at the Saitama Institute of Public Health from January to December 2020. Methods We performed pathogen retrieval using multiplex real-time polymerase chain reaction on samples from patients with acute respiratory diseases who tested negative for SARS-CoV-2 in Saitama in 2020 and analysed the results by age and symptoms. Results There were 1530 patients aged 0–104 years (1727 samples), with 14 pathogens detected from 213 patients (245 samples). Most pathogens were human metapneumovirus (25.4%, 54 cases), rhinovirus (16.4%, 35 cases) and Mycoplasma pneumoniae (13.1%, 23 cases). Human metapneumovirus, human coronavirus (but not NL63) and M. pneumoniae were detected in almost all age groups without any significant bias. Seasonal human coronaviruses, human metapneumovirus, M. pneumoniae and several other pathogens were detected until April 2020. Discussion Multiple respiratory pathogens were circulating during 2020 in Saitama, including SARS-CoV-2 and influenza viruses. We suggest introducing a system that can comprehensively monitor the regional prevalence of all viruses that cause acute respiratory infections.

the detection of various respiratory pathogens in samples from patients who tested negative for SARS-CoV-2 at the Saitama Institute of Public Health from January to December 2020.

Sample selection
Samples sent to the Saitama Institute of Public Health from 30 January to 31 December 2020 that tested negative for SARS-CoV-2 were included in the study.These comprised nasal, pharyngeal and nasopharyngeal swabs; nasal discharge; tracheal aspirate; alveolar lavage fluid; and sputum from people suspected to have COVID-19.As suspected influenza cases are usually confirmed via antigen testing at the clinical site and only positive samples are sent to public health reference laboratories, such specimens were assumed to contain influenza viruses and were excluded. 13e cases' symptoms and age and the date of sample collection were recorded on the laboratory forms collected with the samples.Samples from cases among children aged <15 years were included if they had at least one symptom of fever, upper respiratory tract infection or lower respiratory tract infection (LRTI) reported on the laboratory form; samples from cases aged ≥15 years were included if they had at least one symptom of LRTI reported on the laboratory form.
The number of pathogens detected was tabulated by sample collection date.Cases were divided into three age groups for evaluation, namely paediatric (<15 years), intermediate (≥15 years to <65 years), and elderly people (≥65 years), and the presence of LRTI was assessed in each group.

Pathogen detection procedures
RNA was extracted from specimens using an automated nucleic acid extraction system (EZ1 Advanced XL; QIAGEN, Venlo, Netherlands).Influenza A and B viruses, rhinovirus, adenovirus, enterovirus, human Parechovirus, human metapneumovirus, seasonal human coronaviruses (OC43, 229E, HKU1 and NL63), parainfluenza virus types 1-4, human respiratory syncytial virus (RSV), human bocavirus and Mycoplasma pneumoniae were detected using a multiplex real-time reverse transcriptionpolymerase chain reaction (rRT-PCR) kit (FTD Respiratory Pathogens 21 assay; Siemens Healthcare, Erlangen, Germany).If the samples were positive for influenza virus or RSV, the type or lineage was determined by rRT-PCR.If samples were positive for adenovirus, enterovirus or human Parechovirus, genotyping was performed using Sanger sequencing.

Detection of multiple pathogens
Two different pathogens were detected in 16 cases and three different pathogens were detected in one case (Table 1).
RSV was detected in seven cases (3.3% of 213): RSV-A in four cases (1.9%), RSV-B in two cases (0.9%), and one case could not be typed.Parainfluenza viruses were detected in four cases (1.9%), all type 4. Enterovirus was detected in two cases (0.9%), and coxsackievirus group A type 4 and coxsackievirus group B type 3 were detected in one case each (0.5% each).Human Parechovirus was detected in two cases (0.9%), both of which could not be typed.Influenza virus (B/Victoria lineage) was detected in one case (0.5%).

Classification by age group and symptoms
Based on classifications by age group and the presence of LRTI, the positivity rate observed in the paediatric (2)  transmission might have changed the circulation of seasonal infectious diseases in various regions, 5-8 and the COVID-19 pandemic itself might have suppressed the spread of other respiratory viruses. 14e detection of non-SARS-CoV-2 respiratory pathogens in children suggests that other viruses -such as rhinovirus, adenovirus and human bocavirus -should also be considered in the differential diagnosis of upper respiratory tract infections in children.Differences in viral stability between non-enveloped and enveloped viruses, such as seasonal human coronaviruses and human metapneumovirus, may affect differences in detection. 15Additionally, non-enveloped viruses have been detected in paediatric common in children with LRTI and in the intermediate age group.Although a degree of difference was observed in the positivity rate between the elderly and intermediate age groups, there was no marked difference in the pathogens detected, except M. pneumoniae.

DISCUSSION
We detected a variety of pathogens in samples from patients who had acute respiratory symptoms but had tested negative for SARS-CoV-2 in 2020 in Saitama, Japan.Public health and social measures implemented to prevent SARS-CoV-2 Although weekly reports of the viruses isolated and the detection of cases of upper and lower respiratory inflammation in Japan indicated that respiratory infections spread throughout 2019, 17,18 the decrease in the number of pathogens detected after June 2020 can be partly attributed to the decline in received at the public health laboratory.The Ministry of Health, Labour and Welfare issued a notice on 2 June 2020 allowing PCR testing of saliva samples for SARS-CoV-2, 19 after which the number of respiratory tract samples sent to our laboratory drastically decreased.
In addition, when a patient suspected of having COVID-19 tested negative for SARS-CoV-2, the need for further pathogen testing was determined by the examining doctor.y testing patients with suspected COVID-19 for other viruses that cause acute respiratory infections, we have provided a summary of infections caused by other viruses with similar symptoms.Critical surveillance gaps may be filled by having a more systematic process through which public research institutions such as ours can test samples from cases with influenza-like illness and acute respiratory infections to provide information about prevalence, contagiousness and severity of the disease. 23We propose there is a need to introduce a system that can comprehensively monitor the regional prevalence of all viruses that cause acute respiratory infections, and we hope that the results of this study will be used as a resource to improve surveillance.

Fig. 1 .
Fig. 1.Number of individual respiratory pathogens detected and positivity rate in samples that tested negative for SARS-CoV-2, by month, Saitama, Japan, 2020 a

Table 1 . Cases with multiple respiratory pathogens detected in samples that tested negative for SARS-CoV-2, Saitama, Japan, 2020
LRTI: lower respiratory tract infection; nt: not typed.

Table 2 . Number of cases, number of samples and positivity rate for respiratory pathogens among cases that tested negative for SARS-CoV-2, by age group, Saitama, Japan, 2020Table 3 . Number of positive cases and number of samples of respiratory pathogens from cases that tested negative for SARS-CoV-2, by age group and pathogen, Saitama, Japan, 2020
Columns do not add up to the total as multiple pathogens were detected in some cases and samples. a

Table 4 . Number of cases, number of samples and positivity rate for respiratory pathogens among people who tested negative for SARS-CoV-2, by age group and presence of lower respiratory tract infection, Saitama, Japan, 2020 patients
16d are believed to circulate in immunologically susceptible age groups, raising concerns about outbreaks in the future when nonmedical interventions, such as maskwearing, are lifted. 5-8asonal human coronaviruses have been reported as being more prevalent during winter and early spring;16however, in this study, they were not detected during winter in the second half of 2020.